A novel combinatorial technique for simultaneous quantification of oxygen radicals and aggregation reveals unexpected redox patterns in the activation of platelets by different physiopathological stimuli

Figure 1.. Oxygen radical generation by platelets activated by physiological stimuli collagen and thrombin. Washed human platelets were prepared as described. Collagen was tested at concentrations ranging from 1 to 30 μg/mL (A and C), while thrombin was tested from 0.03 to 1 unit/mL (B and D). Electron paramagnetic resonance (EPR) was used to measure oxygen radicals formation as 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5-tetram-ethylpyrrolidine (CMH) oxidation rates [32.7 and 37.3 attomoles per platelet per minute (min), respectively] (A and C) and aggregation was simultaneously assessed by turbidimetry (B and D). For either technique, representative examples are shown in top panels, while quantification is shown in the bottom panels. Aggregation curves up to 5 min are shown, while EPR resonance readings were taken after 10 min of stimulation. Examples of EPR traces and aggregation curves are representative of 4 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test. *P<0.05 compared to resting platelets. N=4 for (A-D).

Figure 2.. NOX1 is specifically involved in platelet activation by collagen but not thrombin. 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5-tetramethylpyrrolidine (CMH) was utilized for the detection of oxygen radicals generated by platelets. 10 μg/mL collagen (A) or 0.1 unit/mL thrombin (B) were tested. 10 μM of NoxA1ds abolished the electron paramagnetic resonance (EPR) response measured in the presence of collagen. The scrambled peptide at the same concentration (scNoxA1ds) was used as a negative control. Interestingly, the inhibition of NOX1 by NoxA1ds also inhibited collagen-dependent (C), but not thrombin-dependent (D) platelet aggregation. Aggregation curves for up to 5 minutes (min) are shown, while EPR resonance readings were taken after 10 min of stimulation. Examples of EPR traces and aggregation curves are representative of 3 or more independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test for EPR. *P<0.05 compared to resting platelets or t-test for aggregation. *P<0.05 compared to scrambled control. N=3 for (A-D).

Figure 3.. NOX2 is activated by both collagen and thrombin, but essential only for platelet aggregation induced by thrombin. 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5-tetramethylpyrrolidine (CMH) was utilized for the detection of oxygen radicals generated by platelets. 10 μg/mL collagen (A) or 0.1 unit/mL thrombin (B) were tested. 10 μM of Nox2ds-tat inhibited the electron paramagnetic resonance (EPR) response measured in the presence of either collagen or thrombin, although the collagen-dependent response remained significantly higher than resting levels of oxygen radical formation. The scrambled peptide at the same concentration (scNox2ds-tat) was used as a negative control. Interestingly, the inhibition of NOX2 by Nox2ds-tat also inhibited thrombin-dependent (D), but not collagen-dependent (C) platelet aggregation. Examples of EPR traces and aggregation curves are representative of 3 or more independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test for EPR. *P<0.05 compared to resting platelets or t-test for aggregation. *P<0.05 compared to scrambled control. N=4 for (B and D), n=5 for (A), and n=7 for (C).

Figure 4.. NOX1 is the main source of superoxide anions in human platelet response to collagen, while NOX2 is the main source of thrombin-dependent reactive oxygen species (ROS). The activation of NOX1 (A) and NOX2 (B) was assessed by co-immunoprecipitation with their canonical activating and organizing subunits NOXA1 and p47phox, respectively. 400 μL of platelet suspension (4×108 platelets/mL) were stimulated with 10 μg/mL collagen or 0.1 unit/mL thrombin or vehicle solution (Tyrode’s buffer) for 10 minutes (min) before gentle cell lysis (NP40 buffer). Specific NOX1 or NOX2 antibodies and Protein A/G were used to immunoprecipitate the NOX complexes (which by extension should include their regulatory subunits after activation). The immunoprecipitates were tested by immunoblotting using NOX1, NOXA1, NOX2 or p47phox antibodies (as indicated). The data are representative of 4 independent experiments. The functional role of NOX1 and NOX2 in collagen-dependent platelet activation was assessed in a whole blood flow assay (C and D). Platelets were stained with DiOC6 as described and the Bioflux platform (Fluxion, San Francisco, CA, USA) was utilized to assess the thrombus formation induced by…

Figure 5.. NOX1-and NOX2-dependence of collagen and thrombin aggregation and superoxide generation tested in transgenic mice. Platelets were isolated for wild-type (C57BL6/J), NOX1−/− or NOX2−/− mice exsanguinated via intracardiac puncture and resuspended at 2×108 platelets/mL density. Platelets were stimulated with 3 μg/mL collagen (A) or 0.1 unit/mL thrombin (C). Aggregation (left) and superoxide anion formation (right) were measured as described for 5 and 10 minutes (min), respectively. (B) The functional role of NOX1 and NOX2 in collagen-dependent platelet activation was also assessed in a whole blood flow assay. Platelets were stained with DiOC6 and the Bioflux platform (Fluxion, San Francisco, CA, USA) was utilized to assess the thrombus formation induced by collagen under physiological flow (1,000 sec-1). Images were taken at 10 min of flow and are representative of 4 independent experiments. They were quantified by assessing the surface area coverage by platelets with Image J. Data are representative of 4 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test. *P<0.05. N=4 for (A-C).

Figure 6.. NOX1 and NOX2 are required for the induction of superoxide anion formation and platelets activation by oxidized low density lipoprotein (oxLDL). Oxygen radical generation (A) and platelet aggregation (B) in response to oxLDL were measured as described. 10 μM of scrambled NoxA1ds (scNoxA1ds), NoxA1ds, scrambled Nox2ds-tat (scNox2ds-tat), Nox2ds-tat or 100 unit/mL of PEG-SOD were pre-incubated 10 minutes (min) before platelet stimulation with 50 ng/mL oxLDL. Examples of electron paramagnetic resonance (EPR) traces and aggregation curves are representative of 4 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test. *P<0.05. N=3 for (B) and n=4 for (A). The effect of oxLDL as platelet modulator was investigated by traditional aggregometry (C and D). Pre-incubation with 50 ng/mL oxLDL for 10 min was followed by stimulation with 3 μg/mL collagen (C) or 0.03 unit/mL thrombin (D). In order to test the dependence of the modulatory effect of oxLDL on NOX1 and NOX2, 10 μM scrambled NoxA1ds (scNoxA1ds), NoxA1ds, scrambled Nox2ds-tat (scNox2ds-tat), Nox2ds-tat were pre-incubated (10 min before addition of oxLDL). Aggregation curves are…

Figure 7.. NOX1 and NOX2 are required for the induction of superoxide anion formation and platelet activation by amyloid peptide β 1-42. Oxygen radical generation (A) and platelet aggregation (B) in response to amyloid peptide β 1-42 (Aβ1-42) were measured as described. 10 μM of scrambled NoxA1ds (scNoxA1ds), NoxA1ds, scrambled Nox2ds-tat (scNox2ds-tat), Nox2ds-tat or 100 unit/mL of PEG-SOD were pre-incubated 10 minutes (min) before platelet stimulation with 20 μM Aβ1-42. Examples of electron paramagnetic resonance (EPR) traces and aggregation curves are representative of 4 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test. *P<0.05. N=3 for (A and B). The effect of Aβ1-42 as platelet modulator was investigated by traditional aggregometry (C and D). Pre-incubation with 20 μM Aβ1-42 for 10 min was followed by stimulation with 3 μg/mL collagen (C) or 0.03 unit/mL thrombin (D). In order to test the dependence of the modulatory effect of Aβ1-42 on NOX1 and NOX2, 10 μM scrambled NoxA1ds (scNoxA1ds), NoxA1ds, scrambled Nox2ds-tat (scNox2ds-tat), Nox2ds-tat were pre-incubated (10 min before addition of Aβ1-42). Aggregation curves are…

Figure 8.. Experiments on transgenic mice suggest that NOX1 and NOX2 are required for the effect of amyloid peptide β 1-42 and oxidized low density lipoprotein (oxLDL). Platelets from wild-type, NOX1−/− or NOX2−/−mice were stained with DiOC6 and the Bioflux platform (Fluxion, San Francisco, CA, USA) was utilized to assess the thrombus formation induced by collagen under physiological flow. (A) Ibidi Vena8+ flow chambers were coated with 0.05 mg/mL collagen and whole blood was treated with 50 ng/mL native LDL (nLDL) or oxLDL. (C) Ibidi Vena8+ flow chambers were coated with 20 μM Aβ1-42 or scrambled control peptide (ScAβ1-42). The shear rate utilized was 1,000 sec-1, which leads to thrombus formation. Images were taken at 10 minutes (min) of flow and are representative of 4 independent experiments. Images were quantified by assessing the surface area coverage by platelets with Image J. Data are representative of 4 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test. *P<0.05. N=4 for (A and C). Aggregation experiments were performed by pre-treating platelets with 50 ng/mL native LDL (nLDL) or oxLDL (B) or 20 μM Aβ1-42 or scrambled control…